The present invention relates generally to the field of electrophoresis. More particularly, but not by way of limitation, the present invention relates to agarose gel electrophoresis in which the direction of the electric field which is impressed upon the sample may be changed either continuously or discontinuously and periodically by rotating the gel during electrophoresis.
The primary purpose for which the present invention was developed was the fractionation of concatemers of T7 DNA. However, the apparatus and method of the present invention are useful in the fractionation of other large DNA molecules and are broadly applicable to any particle that forms either a random coil or a flexible rod.
Although linear, double-stranded DNA is a spherically symmetric random coil in the absence of a gel, the sieving of this DNA during gel electrophoresis suggests that the DNA elongates and migrates end-first during electrophoresis, a process called reptation. Apparently because of reptation, there is a progressive loss in resolution by molecular weight of linear DNA as both the molecular weight and voltage gradient increase.
The detection, isolation, characterization and quantitation of large open circular DNAs are currently of major importance to research and practice concerning both tumor formation and the development of drug-resistant transformed cells. But, discriminating open circular DNA from linear DNA and fractionating open circular DNA by length present problems when known electrophoresis techniques are used. During agarose gel electrophoresis with an electrical field constant in space and time, open circular DNA will eventually completely stop moving as the voltage gradient is raised. This arrest of DNA occurs more easily as the DNA gets longer and is thought to be the result of threading of the DNA by projections from the gel.
The procedures in known electrophoresis techniques with variable fields have included two orthogonally-oriented sets of vertical electrodes that are alternately energized in a horizontal electrophoresis apparatus. More recently, alternate directions of electrophoresis for DNA random coils have been separated by an angle (.psi.) such that 90&lt;.psi..ltoreq.180.degree.. It will be understood that .psi. stands for the angle between successive applications of the electric field as viewed from the gel medium, whether this angle is the result of varying the electric field relative to a stationary gel or the result of rotating the gel relative to a single, stationary electric field. For values of .psi. other than 180.degree., either a separate apparatus has to be constructed for each .psi., or procedures of voltage clamping have to be used to change .psi.. To avoid the voltage gradient's nonuniformity at different positions in the gel, a problem that plagued initial procedures, either voltage clamping procedures or an altered electrode arrangement with a laterally buffer-exposed vertical gel have been used.
It is known that by changing the direction of electrical fields with two sets of electrodes, larger DNA molecules can be fractionated than by unidirectional electrophoresis. Schwartz et al., in Separation of Yeast Chromosome-Sized DNAs by Pulsed Field Gradient Gel Electrophoresis, Cell, Vol. 37, 67-75, May 1984, describe a system which utilizes perpendicularly oriented, non-uniform, alternately pulsed electrical fields. Using such non-uniform electrical fields, however, results in a distortion of test results, i.e., deformation of bands, and curvature of the path of travel of DNA in the gel, since the forces applied to particles being separated are different for the two directions.
By use of asymmetric times, Carle et al., in Electrophoretic Separations of Large DNA Molecules by Periodic Inverversion of the Electrical Field, Science, Vol. 232, 65-68, 1986, used a .psi. of 180.degree. for a procedure referred to as Field Inversion Gel Electrophoresis (FIGE). However, when using FIGE, length dispersion of the DNA is less regular, length-mobility inversions occur and the length upper limit is only about 1,000 kb. Chu et al., in Separation of Large DNA Molecules by Contour-Clamped Homogeneous Electric Fields, Science, Vol. 234, 1,582-1,585 (1986), changed .psi. to 120.degree. and improved field homogeneity by contour clamping a hexagonal device which is otherwise similar to that of Schwartz et al.
Southern et al., in A Model for the Separation of Large DNA Molecules by Crossed Field Gel Electrophoresis, Nucleic Acids Research, Vol. 15, No. 15, 5925-5943 (1987), describe an apparatus and method for performing rotating gel electrophoresis (RGE). The apparatus comprises a turntable for the gel driven by a DC motor which is coupled with the turntable through a magnetic drive. On receiving a pulse from a timer, the motor drives the turntable round until it is stopped by a microswitch. At the next pulse, a relay reverses the polarity of the supply to the motor which is driven in the opposite direction until it meets a second microswitch. The microswitches are thrown by pins set in the spindle of the magnetic drive and the pin positions can be altered to alter the angle of the turn. The apparatus allows variation in the angle of the field to the gel, and in the voltage and duration of the DC to the electrodes. The apparatus and method of Southern et al. does not allow for modes of operation with .psi..gtoreq.360.degree. having continuous changes of angle as provided by the present invention. Mention, without description, of the device in Southern et al. was made in Anand, Pulsed field gel electrophoresis: A technique for fractionating large DNA molecules, Trends In Genet., Vol. 2, No. 11, 278-283, November, 1986.
The general advantages of rotating gel electrophoresis (RGE), in comparison to known electrophoresis methods are: (a) Electrical fields are as uniform as they are during normal electrophoresis, without the necessity for any additional procedures such as voltage clamping; (b) there is no restriction on .psi.; (c) the deformation of bands is no greater than it is during normal electrophoresis; (d) modes with continuous change of angle can be used and (e) gradients of pH are suppressed by buffer tanks (26 in FIG. 1) with a cross-sectional area greater than that of the gel and superposed buffer. In the absence of such buffer tanks, pH gradients are more difficult to suppress by circulation of buffer. For instance, with the apparatus described by Schwartz et al., but not the apparatus used here, electrophoresis using 0.05M sodium phosphate 0.001M MgCl.sub.2 resulted in the formation of lines of precipitation in the agarose gel, presumably caused by local alkalinization of the phosphate. This precipitation is hard to suppress by circulation. Because double-stranded DNA does not detectably titrate hydrogen ions between pH's 5 and 9, control of pH for double-stranded DNA is less important than it is for protein-containing particles such as the viruses characterized by agarose gel electrophoresis.